During nervous system development, axons are guided through a chemically and physically complex environment in response to repulsive and attractive cues. Accurate axon pathfinding is critical for the establishment of functional neural networks. The elucidation of guidance mechanisms will provide valuable insight into the pathogenesis and treatment of neurodevelopment and neurodegenerative diseases. The objective of this proposal is to test the hypothesis that signaling through the metabotropic 3- aminobutyric acid (GABA) receptor, GABAB reduces axonal responsiveness to repellent guidance cues and that this activity is essential for proper axon guidance in vivo. The role of GABAB receptors in axon guidance will be explored in vivo using the zebrafish retinal projection as a model and in vitro using primary neuronal cultures derived from embryonic chick retinal ganglion cells (RGCs). The repellent Slit2, which binds the Roundabout-2 (Robo2) receptor, is expressed in bands perpendicular to the midline and is believed to determine the location of the optic chiasm via surround repulsion. At the chiasm, axons must travel through areas of Slit2 expression. GABAB activation may be a mechanism through which sensitivity to Slit2 is reduced, permitting crossing despite the presence of a repulsive signal. This proposal consists of three aims. Aim1 is to characterize the expression patterns of GABA and GABAB receptors during zebrafish development using immunohistochemistry and in situ hybridization in whole embryos. Transgenic zebrafish, in which retinal ganglion cells are labeled with green fluorescent protein, will be used to identify the retinal projection. Aim2 is to determine if GABAB signaling is necessary for proper axonal pathfinding in vivo using two independent methods: expression of a dominant negative GABAB receptor (R1C) in RGCs using the UAS-GAL4 system and antisense morpholino knockdown. Aim3 is to determine if GABA/GABAB activity reduces sensitivity to Slit2 by acting as an antirepellent or by activating an attractive signaling pathway. This will be addressed in vitro by performing growth cone turning assays with chick RGCs and in vivo by expressing R1C in mutant zebrafish with either reduced or no Slit/Robo signaling. The zebrafish astray gene encodes Robo2. Both null and hypomorphic alleles result in guidance errors in the retinal projection. If GABAB signaling reduces sensitivity to Slit2, then loss of GABAB signaling should increase sensitivity such that guidance errors due to reduced Slit/Robo signaling are rescued. If GABA/GABAB signaling produces an antirepellent response, then its ability to rescue guidance errors will depend on the presence of Slit/Robo signaling and expression of R1C would rescue defects in the astray hypomorphs but not the null zebrafish. Experiments involving zebrafish will be analyzed via confocal microscopy of whole embryos. PUBLIC HEALTH RELEVANCE: Many of the molecules that are involved in axon guidance have been linked to neurological diseases such as mental retardation, epilepsy, dyslexia, autism, Parkinson's, and Amyotrophic lateral sclerosis (reviewed in Yaron and Zheng, 2006;Anitha et al., 2008;Lesnick et al., 2008). By investigating the role the GABAB receptor plays in axon guidance, we may elucidate normal developmental processes and provide valuable insight into the pathogenesis and treatment of neurodevelopmental and neurodegenerative diseases.